Globally, an estimated 16.2% of the human population is infected with HSV-2 including >17% of the U.S. adult population. Genital herpes is associated with an increased risk of HIV acquisition and transmission of HSV-2 infection can cause neonatal herpes with high infant mortality. HSV-2 infection in the adult population has increased substantially in the past two decades despite the availability of antiviral drugs. The failure of antiviral drugs to prevent the spread of HSV-2 and the sheer magnitude of the public health problem associated with HSV-2 infection indicates a need for a safe and effective vaccine that is not available. As to the protective mechanism, the current consensus of HSV-2 experts is that serum antibody response alone is insufficient to provide protective immunity, primarily based on the disappointing clinical trial results. Therefore, mucosal immunity and T cell responses are considered very important. HSV-2 is a very unique virus able to escape from immune surveillance, and inhibit immune responses in order to successfully infect the host and establish latency. With NIAID-SBIR phase I and II grant funding, we made a significant finding that HSV-2 infected Guinea Pigs (GP) untreated or provided with a reference vaccine (that mimics the HSV-2 vaccine which failed in latest clinical trial) were unable to elicit vaginal IgA. Unlike other HSV-2 vaccine candidates, our mucosal vaccine technology employs a patented heterologous prime-boost strategy consisting of a DNA vaccine prime given intramuscularly (i.m.) and a protein-encapsulated liposome vaccine boost delivered intranasally (i.n.). This two-step heterologous immunization strategy developed as a mucosal vaccine regimen provides a multi-tiered protection with a Th-1 biased, broad and potent cellular and humoral immune response especially mucosal immune response and vaginal protection against primary and latent HSV-2 infection. This novel HSV-2 vaccine shows both prophylactic and therapeutic efficacies particularly at the genital site of the guinea pig models. The company was recently funded by a phase IIB SBIR grant for our HSV-2 vaccine, to support cGMP manufacturing of key vaccine components towards the ultimate goal of IND filing and clinical trials. This new phase I R43 SBIR grant application was developed in response to the Study Section's suggestion of strengthening studies on nasal delivery devices. We plan to conduct bio-distribution, bioavailability studies comparing nasal spray vs. nasal drop delivered HSV-2 gD-Liposome vaccine in New Zealand White rabbits. We also propose to compare the immunogenicity and immune protection efficacies of gD-lip formulation delivered by nasal drop vs. nasal spray in female GPs that will be first primed with our HSV- 2 DNA vaccine. If funded, it would help fill in the gap in the critical pathway of advancing our mucosal HSV-2 vaccine candidate to clinical trials. The net result of these research efforts will be to identify a more efficient nasal delivery device for our mucosal HSV-2 vaccine, and ultimately alleviate pain risk and suffering in the 35- 40 million Americans with genital Herpes.